Mitochondrial diseases caused by mutations in the mitochondrial DNA (mtDNA) are an important source of morbidity and mortality, yet only supportive care is available to affected patients. Mouse models of human disease are instrumental in the developing and testing new therapeutic modalities. So far, the lack of characterized mouse cell lines with mtDNA mutations and technologies to produce such mutations has made impossible routine development of mouse models for the diseases caused by mtDNA mutations. Therefore, the long-term GOAL of this application is to enable routine modeling of human mitochondrial diseases in mice. We propose to achieve this GOAL through (1) refining the methodology developed in the course of preliminary studies; (2) through the use of this methodology to generate a collection of mouse cell lines (3) through the characterization of this collection by sequencing and functional assays, and (4) through the generation of transmitochondrial mice carrying mtDNA mutations generated and characterized in aims 1-3. If successful, these studies will enable routine mouse modeling of mitochondrial disease. Moreover, the outcome of aims 1-3 should generate resources, which will facilitate the structure-function studies of mammalian respiratory complexes. This outcome will also enable generation of a collection of mouse cell lines containing all possible single-nucleotide substitutions in their mtDNA. Candidate: The candidate is a new investigator who received a tenure-track Assistant Professor position in the Department of Cell Biology and Neuroscience, University of South Alabama in 2004. Since then he has authored or co-authored 23 publications in peer-reviewed journals and one book chapter. Most recently, the candidate was invited and wrote a single-author, 20-page review for FEBS journal on the role of mitochondrial DNA in aging process. The candidate's immediate career goal is to secure RO1-level funding for upcoming tenure review. The long-term career goals involve developing a vigorous research program around two main research focused in his lab: the nature of diversity of genotype-phenotype correlations in mitochondrial diseases and mechanisms for the maintenance of mitochondrial DNA integrity. Environment: College of Medicine, University of South Alabama represents a vibrant mitochondrial research environment with 10 faculty members involved in mitochondrial research. Of those, four are senior level researchers (full Professors) and 6 are junior researchers. Both the Chairman and the Vice-Chair of the applicant's Department as well as four junior faculty are mitochondrial researchers. Mitochondrial research in the applicant's Department is supported by three RO1s and one R21. PUBLIC HEALTH RELEVANCE: Animal (mouse) models of human disease are instrumental in developing and testing new therapeutic modalities, yet they are not available for mitochondrial diseases caused by mtDNA mutations. In this application we propose to generate transmitochondrial mice with mtDNA mutations to model human disease.